Cocaine abuse is a major public health problem that directly or indirectly affects most communities and families. There is still no FDA-approved medication specific for cocaine addiction or overdose. Disastrous medical and social consequences of cocaine abuse have made the development of an anti-cocaine medication a high priority. Accelerating cocaine metabolism that produces biologically inactive metabolites via a route similar to the principal cocaine-metabolizing pathway-cocaine hydrolysis catalyzed by human butyrylcholinesterase (BChE) in plasma-is recognized as the most efficient treatment strategy for cocaine overdose and addiction. Since the catalytic efficiency (kcat/KM) of wild-type BChE against the naturally occurring (-)-cocaine is low (kcat = 4.1 min and KM = 4.5 M), we have recently designed and discovered a set of BChE -1 mutants, known as cocaine hydrolases (CocHs), with at least 1,000-fold improved catalytic efficiency against (- )-cocaine compared to wild-type BChE. In vivo and clinical data for the first one of our discovered and patented CocHs demonstrate the promise of enzyme therapy for cocaine abuse. In addition to improved efficacy, our recently designed, discovered, and patented new CocH entities have not only significantly higher catalytic efficiency against (-)-cocaine, but also possess much longer biological half-lives Built on our success in rational design and discovery of the highly efficient CocHs and their long-acting forms (LAFs or CocH-LAFs), this investigation will focus on further optimization, manufacturing process development, formulation development, and preclinical testing of the most promising CocH-LAF as a novel therapeutic candidate for cocaine addiction treatment. The specific aims are: (1) to optimize a promising CocH-LAF which has not only a high catalytic efficiency against (-)-cocaine, but also a long biological half-life; (2) to test feasibility of lage- scale expression of promising CocH-LAF entities; (3) to develop large-scale manufacturing processes for the most promising CocH-LAF selected in Aim 2; (4) to characterize the detailed pharmacology and toxicology profiles of the most promising CocH-LAF in animal models with the CocH-LAF material produced in Aim 3. The most promising CocH-LAF entity developed in this investigation is expected to be highly effective and safe as a novel exogenous enzyme suitable for a monthly dosing schedule for treatment of cocaine addiction in humans. This investigation will make the best possible CocH-LAF entity ready for the cGMP (current Good Manufacturing Practices) protein manufacturing, Investigational New Drug (IND)-enabling studies, and subsequent clinical trials. Thus, this investigation will move a promising therapeutic candidate closer toward FDA approval for cocaine addiction treatment.